Periodontal Disease
Periodontal disease is a chronic inflammatory disease that affects the tissues that support and anchor the teeth, also known as the periodontium. It is caused by the imbalanced interplay between the specific subgingival microorganisms and the host immune and inflammatory response (1). It affects nearly three-quarters of the adult populations and is regarded as one of the most common diseases to human being. The tissues that are involved in periodontal diseases are the gums, which include the gingiva, the periodontal ligament, the cementum, and the alveolar bone (FIG. 1). The gingiva is a pink-colored keratinized mucus membrane that covers parts of the teeth and part of the alveolar bone. The periodontal ligament is the main part of the gums. The cementum is a calcified structure that covers the lower parts of the teeth. The alveolar bone is a set of ridges from the jaw bones (maxillary and mandible) in which the teeth are embedded. The area where periodontal disease is initiated is the gingival sulcus, a pocket between the teeth and the gums.
Infection, inflammation and subsequent host defense and wound healing are all hallmarks of periodontal disease. This disease begins as a mixed bacterial infection in the gingiva surrounding teeth (2). In the healthy mouth, more than 500 species of microorganisms have been found. In periodontal diseases, several potential periodontal pathogens have been studied including Porphyromonas gingivalis, Campylobacter rectus, Actinobacillus actinomcetemcomitans, and Fusobacterium nucleatum, which are considered to represent a significant portion of the pathogenic microbiota. These microorganisms can induce several factors, such as IL-1, IL-6, TNF, as well as enzymes, in host cells which directly or indirectly are thought to cause irreversible tissue destruction including the destruction of the gums, the alveolar bone, the outer layer of the tooth root and eventually leads to tooth loss. Furthermore, serious periodontal disease can lead to bad breath, heart disease and stroke, diabetes, respiratory diseases and premature delivery during pregnancy. There are other pathogenic factors such as smoking/tobacco use, genetics, pregnancy and puberty, stress, medications, diabetes, poor nutrition and other systemic diseases.
Another form of infectious destruction of the alveolar bone, closely resembling periodontitis, namely Periimplantitis, can occur after surgical implantation of an alloplastic material into the jaws. The implantation method is often referred to as osseointegration (3), which entails close contact between the alloplastic material, i.e. the dental implant (often made of titanium), and the living bone. The method is used to restore occlusion subsequent to the loss of natural teeth and is now a standard method for treating edentulism. A principle difference between the osseointegrated dental implant and the natural tooth is the absence of a true periodontium around the implant. While the normal tooth is suspended in a meshwork of collagenous fibers that allows for a physiological mobility of the tooth within the alveolar bone, the dental implant is firmly connected to the bone without intervening soft tissue. Despite this major dissimilarity in attachment to the bone tissue, the pathological changes at teeth and implants during infection share many key features such as infection via biofilm formation and colonization, inflammatory response, as well as immunological defence. Thus, periimplantitis is an inflammatory/infectious process affecting the tissues around an osseointegrated implant in function, resulting in loss of supporting bone. Periimplantitis may lead to complete disintegration and implant loss even if extensive treatment aiming at resolving the periimplant infection has been performed. Periimplantitis also happens as reversible inflammatory/infectious changes of the peri-implant soft tissues without any bone loss. The prevalence of periimplantitis in the soft tissue has been reported in the range of 8-44%, while frequency of periimplantitis in the bone has been reported in the range of 1-19%. The wide ranges for the frequencies seem to be due to differences in defining the entity, at least in part. The frequency of periimplantitis is most likely related to the number of years implants have been worn. Since dental implant treatment was introduced comparatively recently, the numbers will probably increase over the years. Considering the large similarities in the inflammatory response and the immunological defence against infection at teeth and dental implants periimplantitis could be regarded as a form of periodontal disease affecting implanted alloplastic material.
Periodontal disease is an important aspect of general oral health. Oral health refers to the status of health of the oral and related tissues which enables an individual to eat, speak and socialize without active disease, discomfort or embarrassment and which contributes to general well-being. Major indications of oral health include the bacterial flora in the saliva and gum tissue, as well as the tissue necrosis and inflammation in the gum tissue. Oral health is integral to general health and should not be considered in isolation.
Antibiotics and other antimicrobial drugs have been widely used in treatment of infectious diseases since the World War II era. The success of antimicrobials against disease-causing microbes is among modern medicine's great achievements. However, many antimicrobials are not as effective as they used to be. A key factor in the development of antibiotic resistance is the ability of infectious organisms to adapt quickly to new environmental conditions. Over time, some bacteria have developed ways to circumvent the effects of antibiotics. Widespread use of antibiotics is thought to have spurred evolutionarily adaptations that enable bacteria to survive these once so powerful drugs. Ultimately, the increasing difficulty in fighting off microbes leads to an increased risk of acquiring infections in a hospital or other setting. Drug resistance is an especially difficult problem for hospitals harboring critically ill patients who are less able to fight off infections without the help of antibiotics. Therefore, there is an increasing awareness that novel therapeutic strategies are highly needed to improve the infection defense against infection.
Treatment of periodontal disease includes conservative (non-surgical) methods and surgical methods. Conservative treatment consists of deep cleanings known as scaling and rootplaning as well as gingival curettage. This treatment is aimed to remove the biofilm colonizing the affected root surfaces and reestablish an environment where healing can occur. Accompanied with good oral hygiene this will maintain healthy normal gums Surgical periodontal treatment consists of osseous (bone) surgery, gingival/periodontal grafts, gingival flap procedure, frenectomy, gingivectomy, guided tissue regeneration/bone augmentation. However, despite the various therapeutic methods that have successfully improved the treatment of periodontal disease, great challenges in oral health still exist. Such challenging factors include the increasing resistance of oral bacteria against antibiotics, the needs for simpler methods to improve oral health in general, the expensive and tedious dental care procedure, the stressful modern life and the heavier dental burden in under-privileged groups in developed and developing countries. Therefore novel methods of preventing and treating periodontal disease, promoting oral health and improving healing of periodontal wounds are in great needs.
Necrosis
Necrosis is the name given to unprogrammed or accidental death of cells and living tissue. It is less orderly than apoptosis, which are part of programmed cell death. In contrast with apoptosis, cleanup of cell debris by phagocytcs of the immune system is generally more difficult, as the disorderly cell death generally does not send “eat-me” cell signals which tell nearby phagocytes to engulf the dying cell. This lack of signaling makes it harder for the immune system to locate and recycle dead cells which have died through necrosis than if the cell had undergone apoptosis. The release of intracellular content after cellular membrane damage is cause of inflammation in necrosis. There are many causes of necrosis including injury, infection, cancer, infarction, invenomation and inflammation. Severe damage to one essential system in the cell leads to secondary damage to other systems, a so-called “cascade of effects”. Necrosis is caused by special enzymes that are released by lysosomes which are capable of digesting cell components or the entire cell itself. The injuries received by the cell may compromise the lysosome membrane, or may set off an unorganized chain reaction which causes the release in enzymes. Unlike in apoptosis, cells that die by necrosis may release harmful chemicals that damage other cells. Necrosis of biopsy material is halted by fixation or freezing.
Necrosis occurs in certain types of periodontal disease. Necrotizing gingivitis is an inflammatory destructive gingival condition characterized by interproximal necrotic ulcers, spontaneous bleeding, rapid onset of pain and bad odor. Unless properly treated, necrotizing gingivitis has a marked tendency for recurrence and lead to considerable loss of periodontal support.
Currently there are four major therapeutic methods to cure necrosis. The first is surgical, which is the most rapid, and therefore is recommended when large necrotic areas or thick scars are present. The second is mechanical, which includes hydrotherapy, dextranomers and wound irrigation. The third is enzymatical, the enzyme used is mainly collagenase (eg: Santyl), however, the effect is too slow when infection presents; and the fourth is through autolytic method, which is via enzymes in wound fluid but the effect is extremely slow. However, none of the four treatment methods could give a functional and aesthetically satisfactory necrosis removal and tissue remodeling. Therefore, a novel therapeutic strategy is in great need in order to achieve a successful removal of necrosis.
The Plasminogen-Activation System
Plasmin is the key component of the PA system. It is a broad-spectrum protease which has the ability to degrade several components of the ECM including fibrin, gelatin, fibronectin, laminin and proteoglycans (4). In addition, plasmin can convert some pro-matrix metalloproteinases (pro-MMPs) to active MMPs. It has therefore been suggested that plasmin may be an important upstream regulator of extracellular proteolysis (5;6). Plasmin is formed from the zymogen plasminogen through proteolytic cleavage by either of two physiological PAs, tPA or uPA. As plasminogen is present in plasma and other body fluids at relatively high levels, the regulation of the PA system occurs mainly at the level of synthesis and activity of the PAs. Synthesis of the components of the PA system is highly regulated by different factors such as hormones, growth factors and cytokines. In addition, there exist specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is regulated by PAI-1, which inhibits both uPA and tPA, and PAI-2, which inhibits mainly uPA. Certain cells also have a specific cell-surface receptor for uPA (uPAR) that can direct proteolytic activity to the cell surface (8;9).
Plasminogen is a single-chain glycoprotein consisting of 790 amino acids with a molecular mass of approximately 92 kDa (7;8). Plasminogen is mainly synthesized in the liver and is abundant in most extracellular fluids. In plasma the concentration of plasminogen is approximately 2 μM. Plasminogen therefore constitutes a large potential source of proteolytic activity in tissues and body fluids (9;10). Plasminogen exists in two molecular forms: Glu-plasminogen and Lys-plasminogen. The native secreted and uncleaved form has an amino-terminal (N-terminal) glutamic acid and is therefore designated Glu-plasminogen. However, in the presence of plasmin, Glu-plasminogen is cleaved at Lys76-Lys77 to become Lys-plasminogcn. Compared to Glu-plasminogen, Lys-plasminogen has a higher affinity for fibrin and is activated by PAs at a higher rate. These two forms of plasminogen can be cleaved at the Arg560-Val★peptide bond by uPA or tPA, resulting in the formation of the disulphide-linked two-chain protease plasmin (11). The amino-terminal part of plasminogen contains five homologous triple-loops, so-called kringles, and the carboxyl-terminal part contains the protease domain. Some of the kringles contain lysine-binding sites which mediate the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. A novel and interesting finding is that a 38-kDa fragment of plasminogen, consisting of kringles 1-4, is a potent inhibitor of angiogenesis. This fragment is termed angiostatin and can be generated from plasminogen through proteolytic cleavage by several MMPs.
The main substrate for plasmin is fibrin, and dissolution of fibrin is pivotal for prevention of pathological blood clot formation (12). Plasmin also has substrate specificities for several other components of the ECM, including laminin, fibronectin, proteoglycans and gelatin, indicating that plasmin also plays an important role in ECM remodeling (8;13;14). Indirectly, plasmin can also degrade additional components of the ECM through its ability to convert some pro-MMPs to active MMPs, including MMP-1, MMP-2, MMP-3 and MMP-9. It has therefore been suggested that plasmin may be an important upstream regulator of extracellular proteolysis (15). In addition, plasmin has the ability to activate latent forms of certain growth factors (16-18). In vitro, plasmin also cleaves components of the complement system and thereby release chemotactic complement fragments.
The PA system has been suggested to be involved at several stages and by various mechanisms during bacterial invasion (19). A vast number of pathogens express plasmin(ogen) receptors (20;21). Bacteria also influence the secretion of PAs and their inhibitors from mammalian cells (22;23). For instance, production of uPA has been found to be enhanced in cells infected by various bacteria (24). To date, in vivo evidence for a role of plasminogen activation in pathogenesis exists in a few bacteria such as Yersinia pestis, Borrelia, and group A streptococci.
Binding of plasminogen to receptors present on the surfaces of some bacteria convert these bacteria into proteolytic organisms. In Gram-negative bacteria, the filamentous surface appendages form a major group of plasminogen receptors (25;26). In Gram-positive bacteria, surface-bound molecules have been identified as plasminogen receptors (27;28). As a consequence, plasmin can be generated on the surface of microorganisms such as Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Yersinia pestis, and Borrelia burgdorferi, which can lead to a degradation of mammalian ECM. Furthermore, bacterial proteases may also directly activate latent pro-collagenases or inactivate protease inhibitors in human plasma, and thus contribute to tissue damage and bacterial spread across tissue barriers (29;30).
Models of Periodontal Disease and Periodontal Wounds
Models of periodontal disease include spontaneous type and induced type. The periodontal tissue is exposed to a microbe-rich environment. Bacterial invasion and subsequent host defense in the oral cavity occurs constantly and normally remain in balance. Disruption of this host-bacterial balance causes various types of periodontal disease. This could be due to an imbalance between the oral microbiota, alterations in phagocyte function and/or specific immune response. Severe periodontal disease occurs in approximately 2% of US adolescents and in approximately 20% of US adults.
Inducing periodontal disease by certain bacterial species provides defined models for periodontitis. Commonly used periodontal pathogens include Porphyromonus gingivalis, Campylobacter rectus, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum, which are considered to represent a significant portion of the pathogenic microbiota. They possess or can induce in host cells several factors, such as IL-1, IL-6, tumor necrosis factor, surface-associated proteins, fimbriae, vesicles, toxins, and enzymes, which are thought to cause, directly or indirectly, irreversible loss of periodontal supportive tissues.
Periodontal wounds are commonly seen, especially during periodontal surgery. Periodontal wound model can be established by inducing incisional wounds at the gum tissue in the mice. Thereafter the healing pattern of the wounds and the effects of the candidate drug or compounds can be evaluated.
Current method for treating infections such as necrosis as well as periodontal disease have drawbacks as discussed above. Thus, there is still a need in the art for improved strategies and means for treating periodontal disease and improving oral health.